1. Field of the Invention
The present invention relates to improvements in methods and apparatus for sensing and characterizing small particles, such as blood cells or metallic powders, suspended in a liquid medium having electrical impedance per unit volume which differs from that of the particles. More particularly, one aspect of the invention relates to improvements in methods and apparatus for sensing and characterizing such particles, whereby increased sensitivity to particle characteristics other than physical volume is provided. A second aspect of the invention relates to improvements in the non-volumetric sensitivity of methods and apparatus for sensing and characterizing such particles by apparatus operating according to the Coulter principle.
2. Discussion of the Prior Art:
U.S. Pat. No. 2,656,508 to Wallace H. Coulter (the '508 patent) discloses a seminal method for sensing particles suspended in a liquid medium. An exemplary apparatus for implementing such method is schematically illustrated in FIG. 1. Such apparatus comprises a dual-compartment dielectric vessel 6, which defines first and second compartments 6A and 6B separated by a dielectric wall 7. Each of the compartments 6A and 6B is adapted to contain, and is filled with, a liquid medium M. Wall 7 is provided with a relatively large opening 7A, which is sealed by a thin wafer W made of a homogeneous dielectric material. A small through-hole formed in wafer W provides a conduit 10, which constitutes the only operative connection between compartments 6A and 6B. The particles to be sensed and characterized are suspended at an appropriate concentration in liquid medium M and introduced into compartment 6A through a suitable inlet port 8 (or 9) formed therein. A vacuum, provided by an appropriate source in liquid-handling system 13 and operatively coupled to an outlet port 11 suitably formed in compartment 6B, causes the particle suspension to flow from compartment 6A into compartment 6B through conduit 10, discussed in detail below. Each particle in the suspension displaces its own volume of liquid medium M, and conduit 10 provides a consistent reference volume against which that displaced volume may be compared. If the dimensions of conduit 10 and the concentration of particles in the suspension are appropriately selected, particles can be made to transit conduit 10 more or less individually. Conduit 10 then functions as a miniature volumeter, capable under suitable conditions of making sensible the liquid displaced by individual microscopic particles.
To enable convenient sensing of the liquid displacement occasioned by particles transiting conduit 10, liquid medium M is made to have electrical impedance per unit volume which differs from that of the particles. Both aqueous and non-aqueous liquid solutions of a variety of electrolytes have been used as medium M to suspend and carry the particles being characterized through conduit 10. The electrical resistivity p of such liquid media is usually in the approximate range between 30 ohm.multidot.cm and 200 ohm.multidot.cm; e.g., at room temperature the resistivity of a commercial isotonic saline solution (Isoton II, Coulter Corporation) is approximately 61.4 ohm.multidot.cm. The contrast in electrical impedance between particle and medium M thus converts the volume of displaced liquid into a proportional change in the electrical impedance of the liquid column in conduit 10.
The remainder of the apparatus in FIG. 1 forms a two-electrode measurement system responsive to such changes in electrical impedance. Excitation electrodes 15 and 16 are positioned in respective compartments 6A and 6B and operatively connected to a source 17 of electrical current, whereby a nominal electrical current is caused to flow through conduit 10 simultaneously with the particle suspension. Sensing circuitry 18, also operatively connected to excitation electrodes 15 and 16, operates to sense and process pulsations in current between these electrodes. Thus, as individual particles pass through conduit 10, sensing circuit 19 produces an electrical signal pulse having an amplitude which is proportional to the impedance change and therefore characteristic of the particle volume. Additional circuits 20 process the particle signal pulses to provide a count of particles exceeding some particular volumetric threshold. If current source 17 is caused to provide a constant current (so that pulse amplitudes are made insensitive to temperature-induced changes in the electrical conductivity of suspending medium M), the volumetric distribution of the particles may be conveniently characterized through use of multiple-threshold circuitry 21 as described in U.S. Pat. No. 3,259,842 to Wallace H. Coulter et al. Further, if current source 17 is caused to provide at least one alternating-current component at high frequency as discussed in U.S. Pat. No. 3,502,974 to Wallace H. Coulter and W. R. Hogg, an apparent volume reflecting the internal conductivity of biological cells may be similarly characterized. If liquid-handling system 13 comprises a positive-displacement metering system, e.g., such as disclosed in U.S. Pat. No. 2,869,078 to Wallace H. Coulter and Joseph R. Coulter, Jr., such particle counts may be readily displayed or recorded in terms of particle concentration by appropriate devices 22. This method of sensing and characterizing particles, by suspending them in a liquid medium having electrical impedance per unit volume which differs from that of the particles and passing the resulting particle suspension through a constricting conduit while monitoring the electrical current flow through the conduit, has become known as the Coulter principle.
A substantial interphase layer of anions or cations may result when metallic conductors are immersed in a liquid medium comprising ionic species and the ionic medium is constrained to maintain a potential gradient in the vicinity of the conductive material. The predominant ionic type surrounding such conductors depends on the polarity of the potential gradient at the material surface, i.e., on whether electrons enter or leave the material. As noted in the '508 patent, excitation electrodes 15 and 16 develop such concentration polarization layers at their surfaces. At a given temperature, properties of said polarization layers depend on the material of each electrode, the electrolyte, and the local rate of electron exchange (i.e., the current density) through the polarization layer. In the electrode art it is known to minimize local gradients in current density, whereby polarization layers are substantially uniform in thickness and vary from approximately an ionic diameter at very low current densities to perhaps 3.times.10.sup.-5 mm at high current densities. If a given current density is to be maintained between said electrodes, the electrode potentials must be increased to overcome the resistance to electron transfer represented by the polarization layers, and the ions must migrate into medium M. The consequent potential differentials across the thicknesses of the polarization layers are manifested as an overvoltage above the reversible potential at the surface of each electron-exchanging electrode. Thus, the polarization layers act as capacitances in parallel with the charge-transfer resistances, the overvoltages being a logarithmic function of the current density through the two layers. If the potential of electrode 15 or 16 is made to exceed the reversible potential of the particular electrode material in the particular medium M, electrolysis occurs and the consequent bubbles interfere with the ability to sense particles transiting conduit 10.
Electrodes made from metals in the platinum group have comparatively stable overvoltages, and the effects of electrode electrochemistry are further minimized in Coulter apparatus by locating large-area platinum electrodes 15 and 16 away from conduit 10, sometimes in separate electrode chambers electrolytically connected to compartments 6A and 6B. Even so, the electrochemical processes contribute series impedance components and effective back-sources to the electrical equivalent circuit constituted by the electrolytic path between said electrodes and through conduit 10. Consequently, the impedance change occasioned by a particle transiting conduit 10 is superimposed on impedance components originating in the portions of medium M in compartments 6A and 6B. Thus, sensing circuit 19 is preferably AC-coupled with a suitable transient response, whereby the relatively slow impedance variations related to electrochemistry at electrodes 15 and 16 are prevented from interfering with particle-related impedance changes responsible for the desired signal pulses.
Outside the polarization layers ions migrate by a combination of diffusion and ionic drift due to the electric field established between electrodes 15 and 16 by current source 17. Ions migrate through medium M in compartment into the hydrodynamic flow pattern and are carried through Coulter conduit 10 by convective flow. The effective current in the vicinity of conduit 10 is ohmic as determined by the electrical resistivity p of medium M, and the local current density is determined by the geometry along the path of ionic drift. The result is a low resistance due to the portions of medium M in the two compartments of vessel 6, in series with the high resistance formed by the liquid column in conduit 10. Excitation current from current source 17 is limited to the value that causes Joule boiling in conduit 10.
Central to the Coulter principle is volumeter conduit 10, which enables electrical sensing of particle volume by constricting the electric field established in liquid medium M filling vessel 6. Any second path of current conduction between compartments 6A and 6B would act in parallel with the current path through the liquid column in conduit 10 and so by shunting effects would act to decrease the amplitude of signal pulses. For example, if wall 7 or wafer W were to be made of a material less resistive than medium M, intuition suggests that the liquid column in conduit 10 would be bypassed due to its higher resistivity, with excitation current flowing through the more conductive material in proportion to its greater conductivity and area. Consequently, in all Coulter apparatus, functional conduit 10 is formed in a homogeneous dielectric material, the electrical resistivity of which is typically at least 10.sup.9 times that of suspending medium M. In the '508 patent the conduit is a pinpoint aperture formed directly in the wall of a glass vessel, but such conduits proved both difficult to reproduce to the desired precision and prone to damage. An early alternative utilized a separate wafer W, cut from glass capillary tubing and sealed over opening 7A in wall 7 so that the tubing conduit formed conduit 10, but conduit geometry of such wafers was unstable under the glass-fusing methods required for reliable seals. Due to excellent mechanical and dielectric properties, precision ring jewels made of ruby or sapphire are recommended as conduit wafers W in U.S. Pat. No. 2,985,830 to Wallace H. Coulter et al., and such "Coulter wafers" or "aperture wafers" are now extensively used to provide conduit 10. Critical applications may benefit from the superior thermal conductivity of dielectrics such as beryllium oxide or diamond (U.S. Pat. No. 3,771,058 to W. R. Hogg).
A traditional Coulter volumeter conduit 10 (also referred to as a "Coulter aperture") comprises a continuous surface or wall 30 of length L which defines a right cylindrical opening of circular cross-section and diameter D through a homogeneous dielectric wafer W of thickness L and electrical resistivity greater than 10.sup.12 ohm.multidot.cm. As a result, conduit wall 30 surrounding the flows of suspension and excitation current through conduit 10 comprises a single substantially axisymmetric, delimited region of uniformly high resistivity in any longitudinal conduit section. Due to the high resistivity of Coulter wafer W, there is no significant electrical interaction between the material comprised in the wafer and liquid medium M. Consequently, functional attributes of the FIG. 1 apparatus are substantially independent of the specific dielectric material used to form a functional Coulter wafer and the particular medium M used to carry particles through Coulter conduit 10 therein.
Characteristics of signal pulses generated by particles transiting conduit 10 result from complex particle interactions with both the electric field established in liquid medium M by the excitation current and the hydrodynamic field established by the suspending medium carrying the particles through the conduit. For a given particle trajectory through conduit 10, pulse amplitude depends linearly on particle volume and excitation current, but both conduit fields are nonhomogeneous and trajectory-dependent artifactual pulses may be generated. As reviewed in cross-referenced U.S. patent application Ser. No. 08/887,588, considerable remedial art has been developed which minimizes the effects of artifactual pulses on volumetric distributions. Distributional artifacts due to near-wall particle trajectories may be eliminated by causing fluid-handling system 13 to appropriately inject particle suspension through flow director 9 in FIG. 1 (e.g., as described in U.S. Pat. No. 3,810,010 to R. Thom) or reduced by pulse-edit circuit 23 (e.g., as disclosed in U.S. Pat. No. 4,797,624 to H. J. Dunstan et al.). Similar artifacts due to particles on recursing trajectories at the exit of conduit 10 may be eliminated by causing fluid-handling system 13 to provide particle-free sweep-flow via inlet 12 in compartment 6B (e.g., as disclosed in U.S. Pat. No. 3,902,115 to W. R. Hogg et al.) or reduced by pulse-edit circuit 24 (responsive, e.g., to an auxiliary signal as described in U.S. Pat. No. 4,161,690 to M. Feier). Volumetric effects of particle coincidence within conduit 10 may also be limited by correction circuit 25 (e.g., as described in U.S. Pat. No. 3,949,198 to Wallace H. Coulter and W. R. Hogg). When appropriately provided with such facilitating art, Coulter apparatus can generate nearly ideal volumetric distributions for particles transiting conduit 10.
Coulter conduits having diameters ranging between approximately 0.015 mm and 0.200 mm, with conduit length-to-diameter ratios L/D between 0.75 and 1.2, have proven useful for a great variety of particles. For .rho. in ohm.multidot.cm and conduit dimensions expressed in 10.sup.-3 mm, the resistance of the liquid column within such conduits 10 may be estimated as the product of .rho. and the ratio of the length L to the cylindrical cross-sectional area .pi.D.sup.2 /4, EQU R.sub.g =(4.rho..times.10.sup.4)(L/.pi.D.sup.2)ohms. Eq. 1.
Practically, resistances measured between electrodes 15 and 16 are significantly greater than the value calculated by Eq. 1. This is due primarily to effects of the well-known convex equipotentials extending outward from the orifices of conduit 10 and secondarily to the overvoltages of the polarization layers, about 1.1 volts for paired platinum electrodes. The contributions of the two inhomogeneous orifice fields may be approximated by adding a virtual cylinder of diameter D and length 0.2687D to Coulter conduit 10 at both conduit orifices. If this is done and the overvoltages are subtracted, calculated values based on the equivalent uniform cylindrical resistance correspond to measurements of voltage V (in volts) and current I (in amps) made between electrodes 15 and 16: EQU R.sub.c =(V-1.1)/I=(4.rho..times.10.sup.4)(L+0.5374D)/.pi.D.sup.2 ohms. Eq. 2.
In principle, the expected change in resistance .DELTA.R.sub.c between electrodes 15 and 16 due to the passage of a particle through conduit 10 may be similarly estimated according to the resistivity contrast between the particle and the liquid medium M it displaces. Thus, for a dielectric cylindrical particle of length a and diameter d, Eq. 1 predicts a resistance change of EQU .DELTA.R.sub.c.apprxeq.(4.times.10.sup.4)(.rho..sub.p -.rho.)(a/.pi.d.sup.2) ohms, Eq. 3.
where .rho..sub.p is the resistivity of the particle in ohm.multidot.cm and the particle dimensions (a, d) are in 10.sup.-3 mm. For dielectric particles, observed resistance changes are of the order of a few parts per hundred thousand of total resistance. The amplitude of the consequent signal pulse is proportional to the product of .DELTA.R.sub.c and the current I supplied by current source 17. Thus, signal-pulse amplitude should depend on the resistivity contrast (.rho..sub.p -.rho.) and be of a polarity determined by whether the particle is of a dielectric material (.rho..sub.p &gt;.rho.) or a conductive material (.rho..sub.p &lt;.rho.).
However, particles formed of a conductive material also exhibit interphase effects if constrained to maintain a surrounding potential gradient in an electrolyte and so may exhibit a polarization layer and an overvoltage that acts to inhibit charge transfer at the particle surface. Unless the potential gradient represented by the ratio of the overvoltage to the thickness of the polarization layer is exceeded by the axial field gradient in conduit 10, particles of conductive materials are substantially insulated by the polarization layer and behave as though made of dielectric material. These effects are minimized in Coulter apparatus, and it is well known that under typical measurement protocols such apparatus yields positive pulsations substantially identical in amplitude for iso-volumetric spherical particles, irrespective of the electrical resistivity .rho..sub.p of the material forming the specific particle transiting conduit 10. Consequently, Coulter apparatus produces substantially equivalent volumetric distributions for such particles formed of a dielectric material and of a conductive material, and if the two types of particles were admixed and the mixture characterized, a third substantially equivalent volumetric distribution would be produced.
As has been noted, according to aforesaid U.S. Pat. No. 3,502,974 current source 17 is caused to provide a least one alternating-current component at high frequency. For such alternating currents in the range between 20 MHz and 25 MHz the insulating layer formed by a cell membrane is penetrated, and an apparent volume reflecting the internal conductivity of blood cells may be determined. The method is widely used in medical applications of Coulter apparatus and provides significant information that is otherwise unobtainable. At the aforesaid frequencies effects of the polarization layer surrounding conductive particles would be negated, and in principle this method should be applicable to non-biologic particles having significant conductivity. However, displacement currents through the body of the functional Coulter wafer W act to decrease pulse amplitudes, and skin effects further complicate the design of stable implementations. Practically, such instrumentation is complex, and reliable operation at such frequencies is difficult to achieve, particularly for conduit diameters larger than approximately 0.050 mm. The potential demand represented by such material-science applications has not justified commercial instrumentation comprising this complex and costly technology.
It is desirable that simple apparatus for sensing and characterizing particles be provided which offers increased sensitivity to particle characteristics other than physical volume.